The present invention relates to the prevention and treatment of injury and diseases to the liver, biliary tract, bile ducts, gall bladder and related hepatobiliary system. Specifically, the present invention relates to methods for decreasing the action of the RON receptor tyrosine kinase in liver physiology. More specifically, the present invention relates to the use of analogs and antagonists and antibodies for inhibiting the action of the RON receptor tyrosine kinase for the prevention and treatment of liver injury or damage in acute and chronic clinical conditions.
Liver damage may occur in a number of acute and chronic clinical conditions including drug-induced hepatotoxicity, viral infections, vascular injury, autoimmune disease, metabolic disease and blunt trauma.
Acute liver failure comprises a group of severe liver disease with a variety of both known and unknown causative agents. Known potential precipitating agents, for example, include drugs and toxins (e.g. acetaminophen and Amanita mushroom poisoning); viruses (e.g. hepatitis B virus and Epstein Barr virus), and autoimmune hepatitis. Genetic background and/or the presence of underlying liver disease (e.g. preexisting alcoholic steatohepatitis and/or α1-antitrypsin disease) may contribute to the disease process.
At present, there is only one direct therapy for any of the multiple causes of acute liver failure (i.e., the use of N-acetylcysteine in the case of acetaminophen toxicity), and this therapy has not been broadly applicable to other causes of acute liver failure. Supportive care is the mainstay of therapy for most patients. Mortality in adults for all causes of acute liver failure taken together is >60% (even higher in children); the remaining patients progress to orthotopic liver transplantation or, rarely, recover on their own. Few useful markers exist that predict the presence and clinical course of disease, and additional specific therapies to alter the progression of acute liver failure have not been forthcoming (Lee, W. M., (1999) Chapter 35, in Schiff's Diseases of the Liver, 8:879–905).
The pathogenesis of acute liver failure in humans is poorly understood, but an initial understanding is emerging. Kupffer cells (the resident macrophage of the liver) play a prominent role in the disease process, with the release of cytokines and generation of proinflammatory mediators such as tumor necrosis factor α (TNFα) and nitric oxide (NO), both of which can negatively impact the hepatocyte and biliary tree. Endotoxemia is frequent and likely contributes to the activation of Kupffer cells in human disease. (Lee, W. M., (1999), see above). TNFα has been found to be a key mediator of both human and murine forms of acute liver failure (Muto, Y. et al. (1988) Lancet 2:72–74), and expression of TNFα by infiltrating mononuclear cells is associated with increased amounts of apoptotic hepatocytes in acute liver failure (Streetz, K. et al. (2000) Gastroenterol. 119:449–460).
Hepatocyte growth factor-like protein (“HGFL”), also known as macrophage stimulating protein (“MSP”), is a heterodimeric, kringle-containing growth factor belonging to the hepatocyte growth factor family (Bezerra et al. (1999) Protein Science 2, 666–668). HGFL has been found to bind and activate a receptor comprising a heterodimeric transmembrane glycoprotein referred to as “p185RON” or “RON” or stem cell-derived tyrosine kinase. HGFL binds to the RON receptor, which is a tyrosine kinase receptor encoded for by the RON gene (Gaudino, G., et al, (1995) Oncogene 11:2627). Functional characterization of the four kringle and putative non-enzymatically active serine protease-like domains within HGFL/MSP has been performed (Waltz, S. E. et al. (1997) J. Biol. Chem. 272:30256–30537; Wang, M. -H. et al. (1997) J. Biol. Chem. 272:16999–17004). These results suggest that the C-terminal serine protease-like domain (or β chain) of HGFL is required for binding to the RON receptor, and the kringle regions, especially kringle domains 2 and 3, are required for biological function of the HGFL/RON receptor interaction. The majority of mRNA coding for HGFL is expressed in the liver. It is also expressed, at lower levels, in other tissues including the lung, adrenals and placenta. To date, the physiological roles of HGFL in the body have not been fully understood.
Binding of HGFL ligand to RON receptor is expected to lead to receptor homodimerization, thus activating the receptor's tyrosine kinase activity and leading to cross autophosphorylation of tyrosine residues on the adjoining cytoplasmic chain. Phosphorylation of RON receptor then leads to recruitment of SH2-containing effector molecules, such as phospholipase C-γ and phosphatidylinositol-3-kinase (PI-3-kinase; for review see, Leonard, E. J. and Danilkovitch, A. 2000, Adv. Cancer Res. 77, 139–167). Heterodimerization of RON receptor with other tyrosine kinase receptors, such as the Met and erythropoietin receptors, as well as GM-CSF and the common β signal transducer that associates with interleukins-3 and -5, has also become increasingly obvious, and implies multiple roles, and avenues for RON receptor activation (Persons, D. A., et al. (1999) Nat. Genet. 23:159–165; Follenzi, A., et al. (2000) Oncogene 19:3041–3049; Mera, A. et al. (1999) J. Biol. Chem. 274:15766–15774).
A number of biological functions have been described for this ligand/receptor pair, including activation and differentiation of resident peritoneal macrophages (Iwama, A., et al. (1995) Blood 86:3394), inhibition of inducible nitric oxide synthase (iNOS) expression in endotoxin and cytokine-stimulated macrophages (Wang, M. -H., et al. (1994) J. Biol. Chem. 269:14027; Correll, P. H. et al, (1997) Genes Funct. 1:69–83; Muraoka, R. S. et al. (1999) J. Clin. Invest. 103:1277–1285; Waltz, S. E. et al. (2001), J. Clin. Invest. 108, 567–576), and stimulation of proliferation of certain epithelial cell lines and activation of PI-3-kinase activity (Leonard, E. J. and Danilkovitch, A. (2000) Adv. Cancer Res. 77:139–167).
RON activation by HGFL transduces inhibitory signals that block LPS- and IFNγ-induced iNOS expression. RON-mediated inhibition of iNOS expression in macrophages is effected by PI-3-kinase, as illustrated by the effects of transfection of a dominant-inhibitory PI-3-kinase p85 subunit and/or the addition of wortmannin, a specific inhibitor of PI-3-kinase to the response of macrophage to lipopolysaccharide (LPS or endotoxin) and interferon γ (Chen, Y. -Q. et al. (1998) J. Immunol. 161:4950–4959).
Nitric oxide generation occurs in several models of acute liver injury and elevated hepatocellular NO levels can inhibit liver damage in a variety of experimental liver injury models, possibly via inhibition of caspase activities (Torok, N. J. et al., (2000) Hepatology 32:332A, which play key roles in the development of cellular apoptosis. For example, administration of liver-specific NO donors, such as v-PYRRO (Saavedra, J. E., et al. (1997), J. Med. Chem. 40:1947–1954) can markedly blunt the progression of TNFα-induced murine acute liver failure.
The present invention shows that gene-targeted mice lacking the tyrosine kinase-containing cytoplasmic domain of the RON receptor have dramatically altered physiological responses compared to control mice possessing wild type RON receptor. These tyrosine kinase deficient (TK−/−) mice have enhanced lethality to otherwise nontoxic doses of LPS, and macrophage isolated from TK−/− mice have enhanced production of nitric oxide in response to LPS and interferon γ. Dramatic differences in the inflammatory responses in lung and skin injury models have also been noted between control and TK−/− mice (see Waltz, S. E. et al., (2001), J. Clin. Invest. 108, 567–576; McDowell, S. A., et al. (2002) Am. J. Respir. Cell Mol. Biol. 26:99–104).
Little is known about the involvement of the RON receptor in liver physiology, except that it is present in liver tissue.
The present invention now provides for the pharmacological blockage of the hepatocyte growth factor-like protein (“HGFL”) ligand/RON receptor interaction and/or blockage of RON receptor downstream signaling cascades for protective effects towards acute and chronic liver disease, and in particular, acute liver failure.
Therefore, it would be advantageous to have an effective therapy for the prevention and treatment of liver disease. This need exists in any patient population in which chronic or acute liver damage has been induced for example by hepatotoxic compounds, radiation exposure, viral infection, autoimmune disease, and where it is desirable to inhibit the progression of such damage. This need further exists in any patient population at risk of developing liver damage such as in the case of drug overdose, accidental exposure to infected blood samples, or in a clinical setting which includes aggressive chemotherapy, radiation therapy or liver transplantation.